Chemotaxis underlies leukocyte infiltration, recruitment, trafficking and homing, which are not only required for normal immune responses and host defense, but also responsible for many inflammation-related pathological conditions, such as atherosclerosis, ischemia reperfusion-related injuries, angiogenesis, tumorigenesis, etc. Our long term goal is to understand the signaling mechanisms by which chemoattractants regulate leukocyte chemotaxis and their roles in inflammation-related pathophysiological paradigms, particularly atherosclerosis and wound healing. In this proposal, we will continue characterizing chemoattractant signaling mechanisms and their roles in the regulation of directionality and motility, 2 basic components of chemotaxis, using mouse neutrophil as a primary model system. We will use a combination of molecular and cell biological, biochemical, transgenic, proteomic, and siRNA-based functional genomic approaches to accomplish the following specific aims: 1) To investigate the mechanisms by which chemoattractants regulate PTEN and the role of this regulation in chemotaxis. PTEN is a phosphatase that dephosphorylates both protein and phosphoinositide substrates and frequently mutated in many human tumors. Its regulation by extracellular stimuli has not been elucidated. Our preliminary results suggest that small GTPases RhoA and Cdc42 may regulate PTEN localization and activity and that this PTEN regulation is 1 of the key mechanisms for establishing and maintaining directionality. 2) To investigate the role of P-Rexl in chemoattractant-activated signaling and chemotaxis. Our preliminary study of P-Rexl-null leukocytes revealed that P-Rexl is not the primary regulator of Rac activation and F actin formation as previously hypothesized. Nevertheless, its deficiency still resulted in a reduction in F-actin formation albeit at a later phase. We will determine how P-Rexl regulates F actin formation and chemotaxis. 3) To identify and characterize the primary GEF for Rac activation, which should regulate the primary phase of F actin formation. This mechanism should also regulate cell motility, because continuous formation of F actin at the leading edge provides a primary driving force for leukocyte locomotion.